DESCRIPTION (adapted from the applicant's description): Ryanodine receptors (RyRs) are intracellular calcium-releasing channels found in many cell types. They play a key role in regulating cytoplasmic [Ca2+], which controls such basic processes as contraction, secretion, mitosis, and neuroplasticity. RyRs are enriched in striated muscle where they play a central role in excitation-contraction (e-c) coupling, the process by which neuronal depolarization of the muscle membrane leads to release of Ca2+ from the lumen of the sarcoplasmic reticulum into the cytoplasm. A dozen or more mutations have been discovered in skeletal RyRs that together are responsible for two diseases, malignant hyperthermia and central core disease. The potentially important roles of RyRs in heart and brain function (and pathology) are also of current interest. RyRs are tetrameric complexes of net molecular masses greater than 2 million Da, making them the largest ion channels. Further, they interact with other proteins to form complex signaling assemblies, particularly in muscle. High-resolution electron microscopy of purified, frozen RyRs, combined with computerized three-dimensional image reconstruction techniques, offers the methodology for determining the structures of large, multicomponent complexes. This technology will be applied to the following problems: (1) How is the amino acid sequence of the RyR subunit distributed among the various structural domains (at least 11) that have been detected in 3D reconstructions. To accomplish this goal, structures will be determined of RyR:antibody (or Fab) complexes at moderate resolution (2-3nm) using antibodies that are specific to epitopes containing known amino acids within the overall RyR sequence (about 5,000 residues). In collaboration with another laboratory, an alternative molecular-biological approach will be pursued in which recombinant receptors will be prepared that contain epitope tags inserted into surface-exposed regions.(2) Where do natural protein ligands bind on the surface of the RyR? For example, the interaction between the RyR and the dihydropyridine receptor will be investigated, which is thought to be crucial to the mechanism in e-c coupling. (3) How do the three known genetic isoforms of the RyR differ in structure? Each isoform has slightly different properties and physiological roles. For example, only isoform 1 will mediate normal e-c coupling in skeletal muscle. Comparisons of the isoforms in defined functional states (e.g., open, closed, inactivated)in increasing detail will aid in understanding the molecular basis of their different properties and illuminate the structural basis of channel gating.